Metabolic flux profiling of Pichia pastoris grown on glycerol/methanol mixtures in chemostat cultures at low and high dilution rates

1 Department of Chemical Engineering, Universitat Autònoma de Barcelona, 08193-Bellaterra, Spain 2 NMR-laboratory, VTT Technical Research Centre of Finland, PO Box 65, FIN-00014 Helsinki, Finland 3 Department of Chemistry, Universitat Autònoma de Barcelona, 08193-Bellaterra, Spain 4 Department of Chemistry, University at Buffalo, The State University of New York at Buffalo, NY 14260, USA Correspondence Pau Ferrer pau.ferrer{at}uab.cat The metabolic pathways associated with the tricarboxylic acid cycle intermediates of Pichia pastoris were studied using biosynthetically directed fractional 13 C labelling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (1.39 x 10 –5 s –1 and 4.44 x 10 –5 s –1 ) with varying mixtures of glycerol and methanol as sole carbon sources. The results show that, with co-assimilation of methanol, the common amino acids are synthesized as in P. pastoris cells grown on glycerol only. During growth at the lower dilution rate, when both substrates are entirely consumed, the incorporation of methanol into the biomass increases as the methanol fraction in the feed is increased. Moreover, the co-assimilation of methanol impacts on how key intermediates of the pentose phosphate pathway (PPP) are synthesized. In contrast, such an impact on the PPP is not observed at the higher dilution rate, where methanol is only partially consumed. This finding possibly indicates that the distribution of methanol carbon into assimilatory and dissimilatory (direct oxidation to CO 2 ) pathways are different at the two dilution rates. Remarkably, distinct flux ratios were registered at each of the two growth rates, while the dependency of the flux ratios on the varying fraction of methanol in the medium was much less pronounced. This study brings new insights into the complex regulation of P. pastoris methanol metabolism in the presence of a second carbon source, revealing important implications for biotechnological applications. Abbreviations: [ 13 C, 1 H]-COSY, [ 13 C, 1 H] correlation NMR spectroscopy; BDF, biosynthetically directed fractional; cyt, cytosolic; GCV, glycine cleavage pathway; mt, mitochondrial; OAA, oxaloacetate; PPP, pentose phosphate pathway; SHMT, serine hydroxymethyltransferase; TCA, tricarboxylic acid Tables of f -values are available as supplementary data with the online version of this paper. These authors contributed equally to this work.